127These RS libraries in the presence of the cognate tRNATyrCUA were subjected to
a two-step selection process in E. coli (Fig. 3a). The positive selection relied upon
successful suppression of an introduced TAG codon within the reading frame of
the essential chloramphenicol acetyltransferase gene (Wang et al. 2001 ; Fig. 3a).
The growth in the presence of chloramphenicol and the ncAA of interest results in
survival solely of clones containing functional aa-RSs that are able to aminoacyl-
ate the cognate tRNATyrCUA with either the desired ncAA or a canonical amino acid
(Fig. 3a). Elimination of aa-RSs clones recognizing canonical amino acids (i.e. not
specific for the ncAA) relies upon a negative selection, which is most commonly
nonsense suppression of a cell-toxic gene (barnase) that contains introduced amber
codons at permissive sites (Wang and Schultz 2001 ; Chin et al. 2002a; Zhang et al.
2002 ; Fig. 3a). As this selection is performed in the absence of the ncAA, all the
aa-RSs that function with endogenous amino acids are removed from the library
and only clones carrying ncAA-specific RSs survive (Fig. 3a). Multiple rounds of
positive and negative selection are often required to identify an aa-RS that has both
high incorporation efficiency and fidelity for the ncAA of interest. Simplified single
plasmid versions of the screen are available as are alternatives for the negative selec-
tion (Santoro et al. 2002 ; Melancon and Schultz 2009 ). To date, a vast variety of aa-
RSs has been evolved from M. jannaschii Tyr-RS to charge its cognate tRNATyrCUA with
more than 40 structurally different ncAAs (Liu and Schultz 2010 )—highlighting the
flexibility of the enzyme’s active site. Such pairs can exhibit excellent fidelity and
capable yields (shake flask expression of soluble proteins: mg/L range; high-density
fermentation expression of soluble proteins: g/L range; Liu and Schultz 2010 ). De-
spite these notable strengths, the evolved plasticity of the Mj Tyr-RS Tyr binding
site is not limitless thus necessitating the construction of libraries based on other aa-
RS/tRNA pairs. Indeed, several aa-RS/tRNA pairs from Saccharomyces cerevisiae
have been shown to be orthogonal in E. coli (Furter 1998b; Ohno et al. 1998 ; Liu
and Schultz 2006 ), and some orthogonal pairs with hybrid or consensus components
have been adapted for use in E. coli (Kowal et al. 2001 ; Anderson and Schultz 2003 ;
Santoro et al. 2003 ; Anderson et al. 2004 ). Further, orthogonal aa-RS/tRNA pairs
have been identified in the methanogens Methanosarcina barkeri, Methanosarcina
mazei and Desulfitobacterium hafniense that genetically encode Pyrrolysine, the
so-called 22nd canonical amino acid (Srinivasan et al. 2002 ; Krzycki 2005 ). These
Pyl-RS/tRNAPylCUA pairs are discussed separately.
2.2.2 Genetically Encoding non-Canonical Amino Acids in Eukaryotes
Genetic code expansion in eukaryotes holds tremendous promise for the advanced
study of membrane proteins in native cellular environments and for ultimately
revealing molecular mechanisms of cell biology and physiology. Unfortunately,
directed ncAA-RS evolution in mammalian cells is unfeasible due to low trans-
formation efficiencies, slow generation times and comparably low efficiency of
survival-death selection. However, the translation mechanism of the lower eu-
karyote Saccharomyces cerevisiae is conserved with higher eukaryotes, geneti-
Incorporation of Non-Canonical Amino Acids